@ Gaurav:

1. Use an antibody against the protein itself. How many bands do you get? Also, do you even get a band when using antibody against the protein and not his tag. This is important.

2. Are the bands higher or lower than his tagged protein? Higher than normal protein would imply aggregation - this is not a problem to worry about too much.

3. Bands lower than MW, means degradation from the C-terminus if the his tag is on the N-terminus. Depending on the relative intensities of the main expected band and the lower bands one can decide whether to worry about it or not !

4. One must always have protease inhibitors in the buffers. No matter what protein you purify. His-tag affinity chromatography is not a magical technique.!!!! ;-)

@Alejandro: Do you even know whether your protein is soluble or has been discarded with the pellet ?

What did you do to confirm expression of your protein of interest?

Dear Colleagues, to help you some info is required...

- first, you should show your blots, since the discussion is pretty virtual;)

- could be your antibodies recognize artifact bands, to check it E. coli without protein expressed is recommended as an negative control.

- I give 75% chance that you observe the degradation products from the opposite termini to this you tagged. If it is a case double tagging from both termini should help with full length protein production.

@ Gaurav:

1. Use an antibody against the protein itself. How many bands do you get? Also, do you even get a band when using antibody against the protein and not his tag. This is important.

2. Are the bands higher or lower than his tagged protein? Higher than normal protein would imply aggregation - this is not a problem to worry about too much.

3. Bands lower than MW, means degradation from the C-terminus if the his tag is on the N-terminus. Depending on the relative intensities of the main expected band and the lower bands one can decide whether to worry about it or not !

4. One must always have protease inhibitors in the buffers. No matter what protein you purify. His-tag affinity chromatography is not a magical technique.!!!! ;-)

@Alejandro: Do you even know whether your protein is soluble or has been discarded with the pellet ?

What did you do to confirm expression of your protein of interest?

If you see multiple distinct smaller bands in a total lysate directly after harvesting the cells it might be degradation but more like premature end of translation (N-terminal tag) or internal start of translation (C-terminal tag). This happens quote frequently for large proteins with a codon usage that is different from that of E. coli (independent of the using Rosetta strains).

One other (simpler) explanation is unspecific binding of your mono. Have you analyzed non-recombinant E. coli or (better) E.coli expressing another protein in parallel in order to see the band pattern in that lane? If you have the same bands there and the only additional band is one that firs to the expected size than your mono creates the fuzz. There are different qualities out there in the market.

Hi Gaurav,

you cannot expect under your experimental conditions a single band, anti His antibodies are often crappy and/or detect streches of histidines in other proteins. Best you start with a coomassie stain to verify (induced?) expression of your rec. protein. But sometimes the result is not clear because other proteins overlay the rec. one. Then you might do the immunoblot, BUT take only a small amount of extract to reduce unspecific binding. Otherwise, as stated above, an antibody specific to your protein would help. But I would guess then, that the expression of your His-fusion is not fantastic?

Good Luck